Immunization of infants

ABSTRACT

The present invention relates to methods and compositions which may be used to immunize infant mammals against a target antigen, wherein an immunogeniclly effective amount of a nuclic acid encoding a relevant epitope of a desired target antigen is administered to the infant. It is based, at least in part, on the discovery that such genetic immunization of infant mammals could give rise to effective cellular and humoral immune responses against target antigens.

SPECIFICATION

The invention contained herein was funded, at least in part, byNIH-NIAID grant No. GCO #87-009 MI, so that the United States Governmentholds certain rights herein.

1. INTRODUCTION

The present invention relates to methods and compositions which may beused to immunize infant mammals against a target antigen, wherein animmunogenically effective amount of a nucleic acid encoding a relevantepitope of a desired target antigen is administered to the infant.

2. BACKGROUND OF THE INVENTION

A properly operating immune system enables an organism to maintain ahealthy status quo by distinguishing between antigens associated withthe organism itself, which are allowed to persist, and antigensassociated with disease, which are disposed of. Decades ago, Burnetproposed that the immune system's ability to distinguish between “self”and “non-self” antigens results from the elimination of self-reactivelymphocytes in the developing organism (Burnet, 1959, The ClonalSelection Theory of Acquired Immunity, Vanderbilt Univ. Press,Nashville, Tenn.). The phenomenon wherein an organism loses the abilityto produce an immune response toward an antigen is referred to as“tolerance”.

Over the years, a number of observations consistent with the clonalselection theory of tolerance have been documented. For example,genetically non-identical twin cattle, which share a placenta and areexposed to each other's blood cells in utero, fail to reject theallogeneic cells of their sibling as adults (Owen, 1945, Science102:400). As another example, adult rodents that had been injected, atbirth, with hemopoietic cells from a genetically distinct donor rodentstrain were able to accept tissue transplants from that donor strain(Billingham et al., 1953, Nature 172:603; Billingham, 1956, Proc. R.Soc. London Ser. B. 239:44). However, in the early 1980's it was shownthat the injection of minute amounts of antigen (namely animmunoglobulin expressing A48 regulatory idiotype) induced the expansionof helper T cells (Rubinstein et al., 1982, J. Exp. Med. 156:506-521).

The concept of tolerization is associated with the traditional beliefthat neonates are themselves incapable of mounting an effective immuneresponse. It has been generally believed that neonates rely on maternalantibodies (passively transferred via the placenta) for immunity, untilthe neonate begins to synthesize its own IgG anti-bodies (at about 3-4months after birth, in humans; Benjamini and Leskowitz, 1988,“Immunology, A Short Course”, Alan R. Liss, Inc., New York, p. 65). Inspite of the fact that passive immunity still plays a dominant roleuntil 6-8 months after birth, the immune system gradually acquires theability to mount adult-like immune responses.

More recently, several groups have reported findings that dispute thehypothesis that exposure to an antigen in early life disarms the abilityof the immune system to react to that antigen.

Forsthuber et al. (1996, Science 271:1728-1730; “Forsthuber”) suggestthat the impaired lymph node response of so-called “tolerized” mice wasan artifact caused by a technical inability to assess immune function.They reported that neonatal mice, injected with hen egg lysozyme (HEL)in Freund's incomplete adjuvant (“IFA”) according to a protocolconsidered to induce tolerance in adults as well as neonates, displayedan impaired response in the lymph nodes consistent with tolerization.However, the spleen cells of these mice reportedly proliferatedvigorously in response to HEL, a response previously unmeasurable due totechnical limitations. The authors propose that neonatal injection didnot tolerize, but rather induced functional memory cells that weredetectable in spleen but not lymph nodes.

Sarzotti et al. (1996, Science 271:1726; “Sarzotti”) report thatinoculation of newborn mice with a high dose of Cas-Br-M murine leukemiavirus (“Cas”) does not result in immunological unresponsiveness, butrather leads to a nonproductive type 2 response which is likely to havea negative effect on the induction of mature effector cells. Accordingto Sarzotti, clonal deletion of relevant CTL was not observed in miceinfected at birth with a low dose of Cas.

Finally, Ridge et al. (1996, Science 271:1723-1726; “Ridge”) proposesthat previous reports of tolerance induction may have been associatedwith a relative paucity of antigen presenting cells. Ridge observed theinduction of CTL reactivity in neonatal mice injected with antigenexpressed on dendritic cells (which are so-called professional antigenpresenting cells).

The use of nucleic acids as vaccines was known prior to the presentinvention (see, for example, International Application Publication No.WO 94/21797, by Merck & Co. and Vical, Inc., and InternationalApplication Publication No. WO 90/11092). It was not known, however,that such vaccines could be used to induce an immune response, includinghumoral and cellular components, in infant mammals.

3. SUMMARY OF THE INVENTION

The present invention relates to methods and compositions which may beused to immunize infant mammals against a target antigen, wherein animmunogenically effective amount of a nucleic acid encoding a relevantepitope of a desired target antigen is administered to the infant. It isbased, at least in part, on the discovery that such genetic immunizationof infant mammals could give rise to effective cellular (including theinduction of cytotoxic T lymphocytes) and humoral immune responsesagainst target antigen. Moreover, the present invention may reduce theneed for subsequent boost administrations (as are generally required forprotein and killed pathogen vaccines), and may prevent side-effectsassociated with live attenuated vaccines. For instance, the World HealthOrganization recommends waiting nine months after birth beforeimmunizing against rubella, measles, and mumps, in order to avoidundesirable side effects associated with vaccination against thesediseases. Similarly, the World Health Organization recommends waitingtwo months after birth before immunizing children against influenzavirus. In addition to concern over side effects, there is doubt as towhether an effective immune response may be generated using theseconventional vaccines prior to the recommended ages.

4. DESCRIPTION OF THE FIGURES

FIGS. 1A-F. Primary and secondary NP-specific cytotoxicity one monthafter injection of newborn (D-F) or adult (A-C) mice with DNA encodinginfluenza nucleoprotein (NPV1). The percentage of specific lysis wasdetermined in a standard 4-hour ⁵¹Cr release assay for CTL (cytotoxic Tlymphocytes) obtained from newborn or adult animals immunized with NPV1or control DNA and boosted (or not) with live PR8 virus one month aftercompleting the immunization. An additional control group was injectedwith saline and boosted one month later with virus. Spleen cells wereharvested 7 days after boosting and the percentage of NP-specificcytotoxicity was determined immediately (i.e., primary cytotoxicity) orafter incubation for five days with irradiated spleen cells, NP peptide,and IL-2 (i.e., secondary cytotoxicity) as described in Zaghouani etal., 1992, J. Immunol. 148:3604-3609. CTLs were assayed against P815cells coated with NP peptide (5 μg/ml) or infected with PR8 (not shown)or B Lee virus.

FIGS. 2A-B. Limiting dilution assay to determine the frequency ofNP-specific CTL precursors one month after injection of newborn (B) andadult (A) mice with NPV1. Splenocytes harvested 7 days after PR8boosting from newborn and adult mice vaccinated with NPV1 or controlplasmid were incubated in serial dilution (6×10⁴ to 2×10¹splenocytes/well) for 5 days with x-irradiated, PR8-infected splenocytesfrom non-immunized BALB/c mice in the presence of IL-2 (6 units/ml). Theincubation was carried out in 96-well microtiter plates with 24 wellsfor each dilution of effector cells. Cytotoxicity was assessed againstPR8-infected or non-infected P815 cells. Those wells exhibitingpercentage lysis greater than background plus three standard deviationswere regarded as positive.

FIG. 3. Detection of DNA in muscle of BALB/c mice infected with NPV1.Muscle tissue was removed from the site of injection in the rightgluteal muscle of newborns or tibial muscle of adults one month aftercompletion of the vaccination schedule. DNA recovered from the muscletissue on the left flank of each animal served as a control. Thelabeling above each lane indicates the origin of DNA.

FIGS. 4 A-C. Cross-reactive CTLS generated in newborns injected withNPV1. The percentage of specific lysis was determined using a standard⁵¹Cr release assay. Spleen cells were harvested from (A) PR8 immunizedmice; (B) genetically immunized newborns that were immunized one monthlater with PR8 virus and (C) genetically immunized newborns Spleen cellswere cultured for 4 days with irradiated PR8-infected spleen cells, thenassayed in the presence of ⁵¹Cr-labeled P815 cells noninfected orinfected with PR8, A/HK, A/Japan or B lee virus.

FIGS. 5A-F. Survival of genetically immunized newborn and adult micechallenged 1 mo. (A-D) or 3 mo. (E and F) after immunization with1.5×10⁴ TCID₅₀ PR8 virus or 3×10⁵ TCID₅₀ HK virus via aerosol.

FIGS. 6A-B. Kinetics of body-weight loss and recovery in immunized adult(A) or newborn (B) mice challenged with 1.5×10⁴ TCID50 PR8 virus onemonth after completing the immunization.

FIGS. 7A-D. Survival of (A-B) newborn and (C-D) adult mice immunizedwith pHA plasmid encoding hemagglutinin of WSN influenza virus andchallenged with LD₁₀₀ of WSN or PR8 virus, 1 month after immunization.

5. DETAILED DESCRIPTION OF THE INVENTION

For purposes of clarity of description, and not by way of limitation,the detailed description of the invention is divided into the followingsubsections:

(i) compositions for immunization; and

(ii) methods of immunization.

5.1. Compositions For Immunization

The present invention provides for compositions which may be used toimmunize infant mammals against a target antigen which comprise aneffective amount of a nucleic acid encoding a relevant epitope of thetarget antigen in a pharmaceutically acceptable carrier.

Nucleic acids which may be used herein include deoxyribonucleic acid(“DNA”) as well as ribonucleic acid (“RNA”). It is preferable to use DNAin view of its greater stability to degradation.

The term “target antigen” refers to an antigen toward which it isdesirable to induce an immune response, Such an antigen may be comprisedin a pathogen, such as a viral, bacterial, protozoan, fungal, yeast, orparasitic antigen, or may be comprised in a cell, such as a cancer cellor a cell of the immune system which mediates an autoimmune disorder.For example, but not by way of limitation, the target antigen may becomprised in an influenza virus, a cytomegalovirus, a herpes virus(including HSV-I and HSV-II), a vaccinia virus, a hepatitis virus(including but not limited to hepatitis A, B, C, or D), a varicellavirus, a rotavirus, a papilloma virus, a measles virus, an Epstein Barrvirus, a coxsackie virus, a polio virus, an enterovirus, an adenovirus,a retrovirus (including, but not limited to, HIV-1 or HIV-2), arespiratory syncytial virus, a rubella virus, a Streptococcus bacterium(such as Streptococcus pneumoniae), a Staphylococcus bacterium (such asStaphylococcus aureus), a Hemophilus bacterium (such as Hemophilusinfluenzae), a Listeria bacterium (such as Listeria monocytogenes), aKlebsiella bacterium, a Gram-negative bacillus bacterium, an Escherichiabacterium (such as Escherichia coli), a Salmonella bacterium (such asSalmonella typhimurium), a Vibrio bacterium (such as Vibrio cholerae) ,a Yersinia bacterium (such as Yersinia pestis or Yersiniaenterocoliticus), an Enterococcus bacterium, a Neisseria bacterium (suchas Neiserria meningitidis), a Corynebacterium bacterium (such asCorynebacterium diphtheriae), a Clostridium bacterium (such asClostridium tetani), a Mycoplasma (such as Mycoplasma pneumoniae), aPseudomonas bacterium, (such as Pseudomonas aeruginosa), a Mycobacteriabacterium (such as Mycobacterium tuberculosis), a Candida yeast, anAspergillus fungus, a Mucor fungus, a toxoplasma, an amoeba, a malarialparasite, a trypanosomal parasite, a leishmanial parasite, a helminth,etc. Specific nonlimiting examples of such target antigens includehemagglutinin, nucleoprotein, M protein, F protein, HBS protein, gp120protein of HIV, nef protein of HIV, and listeriolysine. In alternativeembodiments, the target antigen may be a tumor antigen, including, butnot limited to, carcinoembryonic antigen (“CEA”), melanoma associatedantigens, alpha fetoprotein, papilloma virus antigens, Epstein Barrantigens, etc.

The term “relevant epitope”, as used herein, refers to an epitopecomprised in the target antigen which is accessible to the immunesystem. For example, a relevant epitope may be processed afterpenetration of a microbe into a cell or recognized by antibodies on thesurface of the microbe or microbial proteins. Preferably, an immuneresponse directed toward the epitope confers a beneficial effect; forexample, where the target antigen is a viral protein, an immune responsetoward a relevant epitope of the target antigen at least partiallyneutralizes the infectivity or pathogenicity of the virus. Epitopes maybe B-cell or T-cell epitopes.

The term “B cell epitope”, as used herein, refers to a peptide,including a peptide comprised in a larger protein, which is able to bindto an immunoglobulin receptor of a B cell and participates in theinduction of antibody production by the B cells.

For example, and not by way of limitation, the hypervariable region 3loop (“V3 loop”) of the envelope protein of human immunodeficiency virus(“HIV”) type 1 is known to be a B cell epitope. Although the sequence ofthis epitope varies, the following consensus sequence, corresponding toresidues 301-319 of HIV-1 gp120 protein, has been obtained:Arg-Lys-Ser-Ile-His-Ile-Gly-Pro-Gly-Arg-Ala-Phe-Tyr-Thr-Thr-Gly-Glu-Ile-Ile(SEQ ID NO:1).

Other examples of known B cell epitopes which may be used according tothe invention include, but are not limited to, epitopes associated withinfluenza virus strains, such as Trp-Leu-Thr-Lys-Lys-Gly-Asp-Ser-Tyr-Pro(SEQ ID NO:2), which has been shown to be an immunodominant B cellepitope in site B of influenza HA1 hemagglutinin, the epitopeTrp-Leu-Thr-Lys-Ser-Gly-Ser-Thr-Tyr-Pro (H3; SEQ ID NO:3), and theepitope Trp-Leu-Thr-Lys-Glu-Gly-Ser-Asp-Tyr-Pro (H2; SEQ ID NO:4) (Li etal., 1992, J. Virol. 66:399-404); an epitope of F protein of measlesvirus (residues 404-414; Ile-Asn-Gln-Asp-Pro-Asp-Lys-Ile-Leu-Thr-Tyr SEQID NO:5; Parlidos et al., 1992, Eur. J. Immunol. 22:2675-2680); anepitope of hepatitis virus pre-Sl region, from residues 132-145(Leclerc, 1991, J. Immunol. 147:3545-3552); and an epitope of foot andmouth disease VP1 protein, residues 141-160,Met-Asn-Ser-Ala-Pro-Asn-Leu-Arg-Gly-Asp-Leu-Gln-Lys-Val-Ala-Arg-Thr-Leu-Pro(SEQ ID NO:6; Clarke et al., 1987, Nature 330:381-384).

Still further B cell epitopes which may be used are known or may beidentified by methods known in the art, as set forth in Caton et al.,1982, Cell 31:417-427.

In additional embodiments of the invention, peptides which may be usedaccording to the invention may be T cell epitopes. The term “T cellepitope”, as used herein, refers to a peptide, including a peptidecomprised in a larger protein, which may be associated with MHC selfantigens and recognized by a T cell, thereby functionally activating theT cell.

For example, the present invention provides for T_(h) epitopes, which,in the context of MHC class II self antigens, may be recognized by ahelper T cell and thereby promote the facilitation of B cell antibodyproduction via the T_(h) cell.

For example, and not by way of limitation, influenza A hemagglutinin(HA) protein of PR8 strain, bears, at amino acid residues 110-120, aT_(h) epitope having the amino acid sequenceSer-Phe-Glu-Arg-Phe-Glu-Ile-Phe-Pro-Lys-Glu (SEQ ID NO:7).

Other examples of known T cell epitopes include, but are not limited to,two promiscuous epitopes of tetanus toxoid,Asn-Ser-Val-Asp-Asp-Ala-Leu-Ile-Asn-Ser-Thr-Lys-Ile-Tyr-Ser-Tyr-Phe-Pro-Ser-Val(SEQ ID NO:8) andPro-Glu-Ile-Asn-Gly-Lys-Ala-Ile-His-Leu-Val-Asn-Asn-Glu-Ser-Ser-Glu (SEQID NO:9, Ho et al., 1990, Eur. J. Immunol. 20:477-483); an epitope ofcytochrome c, from residues 88-103,Ala-Asn-Glu-Arg-Ala-Asp-Leu-Ile-Ala-Tyr-Leu-Gln-Ala-Thr-Lys (SEQ IDNO:10); an epitope of Mycobacteria heatshock protein, residues 350-369,Asp-Gln-Val-His-Phe-Gln-Pro-Leu-Pro-Pro-Ala-Val-Val-Lys-Leu-Ser-Asp-Ala-Leu-IleSEQ ID NO:11; Vordermir et al., Eur. J. Immunol. 24:2061-2067); anepitope of hen egg white lysozyme, residues 48-61,Asp-Gly-Ser-Thr-Asp-Tyr-Gly-Ile-Leu-Gln-Ile-Asn-Ser-Arg (SEQ ID NO:12;Neilson et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:7380-7383); anepitope of Streptococcus A M protein, residues 308-319,Gln-Val-Glu-Lys-Ala-Leu-Glu-Glu-Ala-Asn-Ser-Lys (SEQ ID NO:13; Rossiteret al., 1994, Eur. J. Immunol. 24:1244-1247); and an epitope ofStaphylococcus nuclease protein, residues 81-100,Arg-Thr-Asp-Lys-Tyr-Gly-Arg-Gly-Leu-Ala-Tyr-Ile-Tyr-Ala-Asp-Gly-Lys-Met-Val-Asn(SEQ ID NO:14; de Magistris, 1992, Cell 68:1-20). Still further T_(h)epitopes which may be used are known or may be identified by methodsknown in the art.

As a further example, a relevant epitope may be a T_(CTL) epitope,which, in the context of MHC class I self antigens, may be recognized bya cytotoxic T cell and thereby promote CTL-mediated lysis of cellscomprising the target antigen. Nonlimiting examples of such epitopesinclude epitopes of influenza virus nucleoproteins TYQRTRALVRTGMDP (SEQID NO:15) or IASNENMDAMESSTL (SEQ ID NO:16) corresponding to amino acidresidues 147-161 and 365-379, respectively (Taylor et al., 1989Immunogenetics 26:267; Townsend et al., 1983, Nature 348:674); LSMVpeptide, KAVYNFATM, amino acid residues 33-41 (SEQ ID NO:17);Zinhernagel et al., 1974, Nature 248:701-702); and ovalbumin peptide,SIINFEKL, corresponding to amino acid residues 257-264 (SEQ ID NO:18;Cerbone et al., 1983, J. Exp. Med. 163:603-612).

The nucleic acids of the invention encode a relevant epitope, and mayoptionally further comprise elements that regulate the expression and/orstability and/or immunogenicity of the relevant epitope. For example,elements that regulate the expression of the epitope include, but arenot limited to, a promoter/enhancer element, a transcriptionalinitiation site, a polyadenylation site, a transcriptional terminationsite, a ribosome binding site, a translational start codon, atranslational stop codon, a signal peptide, etc. Specific examplesinclude, but are not limited to, a promoter and intron A sequence of theinitial early gene of cytomegalovirus (CMV or SV40 virus (“SV40”);Montgomery et al., 1993, DNA and Cell Biology 12:777-783). With regardto enhanced stability and/or immunogenicity of the relevant epitope, itmay be desirable to comprise the epitope in a larger peptide or protein.For example, and not by way of limitation, the relevant epitope may becomprised in an immunoglobulin molecule, for example, as set forth inU.S. patent application Ser. No. 08/363,276, by Bona et al., thecontents of which is hereby incorporated in its entirety herein byreference. Alternatively, more than one epitope may be expressed withinthe same open reading frame.

Nucleic acids encoding the relevant epitope and optionally comprisingelements that aid in its expression, stability, and/or immunogenicitymay be comprised in a cloning vector such as a plasmid, which may bepropagated using standard techniques to produce sufficient quantities ofnucleic acid for immunization. The entire vector, which may preferablybe a plasmid which is a mammalian expression vector comprising thecloned sequences, may be used to immunize the infant animal. Sequencesencoding more than one epitope may be comprised in a single vector.

Examples of nucleic acids which may be used according to the inventionare set forth in International Application Publication No. WO 94/21797,by Merck & Co. and Vical, Inc., and in International ApplicationPublication No. WO 90/11092, by Vical, Inc., the contents of which arehereby incorporated in their entireties herein by reference.

Different species of nucleic acid, encoding more than one epitope of oneor more target antigens, may be comprised in the same composition or maybe concurrently administered as separate compositions.

The term “effective amount”, as used herein, refers to an amount ofnucleic acid encoding a relevant epitope of a target antigen, which,when introduced into a infant mammal, results in a substantial increasein the immune response of the mammal to the target antigen. Preferably,the cellular and/or humoral immune response to the target antigen isincreased, following the application of methods of the invention, by atleast four-fold, and preferably by at least between 10-fold and 100-fold(inclusive), above baseline. The immunity elicited by such geneticimmunization may develop rapidly after the completion of theimmunization (e.g., within 7 days), and may be long lasting (e.g.,greater than 9 months). The need for “boosting” in order to achieve aneffective immune response may be diminished by the present invention. Inpreferred embodiments, the effective amount of nucleic acid isintroduced by multiple inoculations (see below).

In specific, nonlimiting embodiments of the invention, nucleic acidencoding between 1-500 picomoles of relevant epitope, preferably between20-100 picomoles of relevant epitope, and more preferably between 40-100picomoles of relevant epitope per gram weight of the infant mammal maybe administered.

The nucleic acids of the invention may be comprised in apharmaceutically acceptable carrier, for example, but not limited to,physiologic saline or liposomes. In specific, nonlimiting embodiments,the concentration of nucleic acid preferably ranges from 30-100 μg/100μl. In certain embodiments, it may be desirable to formulate suchcompositions as suspensions or as liposomal formulations.

5.2. Methods of Immunization

The present invention provides for a method for immunizing an infantmammal against a target antigen, comprising inoculating the mammal withan effective amount of a nucleic acid encoding a relevant epitope of thetarget antigen in a pharmaceutically acceptable carrier.

The term “infant”, as used herein, refers to a human or non-human mammalduring the period of life following birth wherein the immune system hasnot yet fully matured. In humans, this period extends from birth to theage of about nine months, inclusive. In mice, this period extends frombirth to about four weeks of age. The terms “newborn” and “neonate”refer to a subset of infant mammals, which have essentially just beenborn. Other characteristics associated with “infants” according to theinvention include, an immune response which has: (i) susceptibility tohigh-zone tolerance (deletion/anergy of T cell precursors, increasedtendency to apoptosis); (ii) a Th2 biased helper response (phenotypicalparticularities of neonatal T cells; decreased CD40L expression onneonatal T cells); (iii) reduced magnitude of the cellular response(reduced number of functional T cells; reduced antigen-pre-senting cellfunction); and (iv) reduced magnitude and restricted isotope of humoralresponse (predominance of IgM^(high) IgD^(low) B cells, reducedcooperation between Th and B cells).

In specific nonlimiting embodiments of the invention, nucleic acidimmunization may be administered to an infant animal wherein maternalantibodies remain present in detectable amounts. In a relatedembodiment, the pregnant mother may be immunized with a nucleicacid-based vaccine prior to delivery so as to increase the level ofmaternal antibodies passively transferred to the fetus.

The terms “immunize” or “immunization” or related terms refer herein toconferring the ability to mount a substantial immune response(consisting of antibodies or cellular immunity such as effector CTL)against a target antigen or epitope. These terms do not require thatcompletely protective immunity be created, but rather that a protectiveimmune response be produced which is substantially greater thanbaseline. For example, a mammal may be considered to be immunizedagainst a target antigen if the cellular and/or humoral immune responseto the target antigen occurs following the application of methods of theinvention. Preferably, immunization results in significant resistance tothe disease caused or triggered by pathogens expressing target antigens.

The term “inoculating”, as used herein, refers to introducing acomposition comprising a nucleic acid according to the invention into ainfant animal. Such introduction may be accomplished by any means androute known in the art, including intramuscular, subcutaneous,intravenous, intraperitoneal, intrathecal, oral, nasal, rectal, etc.administration. Preferably, inoculation is performed by intramuscularinjection.

The effective amount of nucleic acid is preferably administered inseveral inoculations (that is to say, the effective amount may be splitinto several doses for inoculation). The number of inoculations ispreferably at least one, and is more preferably three.

The success of the inoculations may be confirmed by collecting aperipheral blood sample from the subject between one and four weeksafter immunization and testing for the presence of CTL activity and/or ahumoral response directed against the target antigen, using standardimmunologic techniques.

In specific, nonlimiting embodiments, the present invention may be usedto immunize a human infant as follows. A human infant, at an age rangingfrom birth to about 9 months, preferably at an age ranging from birth toabout 6 months, more preferably at an age ranging from birth to about 1month, and most preferably at an age ranging from birth to about 1 week,may commence a program of injections whereby the infant may be injectedintramuscularly three times at 3-7 day intervals with a compositioncomprising 1-100 manomoles of DNA encoding a relevant epitope of atarget antigen, preferably at a DNA concentration of 1-5 mg/100 μl,wherein the target antigen may by a protein from a pathogen, for examplerespiratory syncytial virus, rotavirus, influenza virus, hepatitisvirus, or HIV virus (see above).

Accordingly, the present invention provides for compositions for use inimmunizing an infant mammal against a target antigen, comprising anucleic acid encoding said target antigen in an amount effective ininducing a cellular (e.g. CTL) and/or humoral immune response.

It is believed that one of the advantages of the methods of theinvention is that mammals immunized by such methods may exhibit a lessertendency to develop an allergy or other adverse reaction after exposureto target antigens. Further, DNA vaccination of infants may reduce therisk of tolerance induction following other vaccination protocols whichrequire successive administration of relatively high doses of antigen.

6. EXAMPLE: INDUCTION OF CELLULAR IMMUNITY AGAINST INFLUENZA VIRUSNUCLEOPROTEIN IN NEWBORN MICE BY GENETIC VACCINATION

6.1. Materials And Methods

Plasmids. The NPV1 plasmid (obtained from Dr. Peter Palese) wasconstructed by inserting a cDNA derived from the nucleoprotein gene ofA/PR8/34 into the BglII site of a mutated pBR322 vector, namelypCMV-IE-AKi-DHFR (Whong et al., 1987, J. Virol. 61:1796), downstreamfrom a 1.96 kb segment of the enhancer, promoter and intron A sequenceof the initial early gene of cytomegalovirus and upstream of a 0.55 kbsegment of the β globin polyadenylation signal sequence as described inUlmer et al., 1993, Science 259:1745. The modified pBR322 vector withoutthe NP sequence (termed the “V1 plasmid”) was employed as a control.PRc/CMV-HA/WSN plasmid (pHA plasmid or WSN-HA plasmid) was constructedby inserting HA of A/WSN/33 (subtype H1N1) strain of influenza virusinto the PRc/CMV mammalian expression vector and donated by Dr. PeterPalese (Mount Sinai School of Medicine). All plasmids were propagated inEscherichia coli and purified by the alkaline lysis method (Id.).

Viruses. The influenza virus strains A/PR8/34 (H1N1), A/HK/68(H3N2),A/Japan/305/57(H2N2) and B Lee/40 were grown in the allantoic cavity ofembryonated hen eggs as described in Kilbourne, 1976, J. Infect. Dis.134:384-394. The A/HK/68 virus adapted to mice was provided by Dr.Margaret Liu (Merck Research Laboratories). The influenza virus strainA/WSN/33 was grown in MDBK cells and purified from supernatants.

Immunization. One month old adult mice were vaccinated with 30 μg ofNPV1, pHA or control plasmid dissolved in 100 μl of physiologic salineby injection into the anterior tibial muscle of the shaved right legusing a disposable 28 gauge insulin syringe that was permitted topenetrate to a depth of 2 mm; three injections with 30 μg DNA werecarried out at three week intervals. Newborn mice were immunized with 30μg of plasmid dissolved in 50 μl of physiologic saline by similarinjection into the right gluteal muscle of Days 1, 3 and 6 after birthof life. Some newborn mice were injected intraperitoneally (“IP”) on Day1 after birth with PR8 or B Lee live virus (5 μg in 0.1 ml saline). Onemonth after completion of the vaccination schedule, some mice wereboosted with live virus in saline at a dose of 1×10³ TCID₅₀ injected ip.

Infection. Mice were challenged via the aerosol route with 1.5×10⁴TCID₅₀ of A/PR8/34 (LD100) or 3.2×10⁵ TCID₅₀ of A/HK/68 (LD₁₀₀ virus) or3×10⁷ TCID₅₀ of A/WSN/33 (LD₁₀₀). Exposure was carried out for 30minutes in an aerosol chamber to which a nebulizer (Ace Glass, Inc.) wasattached via a vacuum/pressure system pump operated at a rate of 35L/min and a pressure of 15 lb/in². Mice were observed once dailypost-infection and their survival was recorded.

Viral Lung Titers. Processing of lung tissue was carried out with atleast three mice from each treatment group as described in as describedin Isobe et al., 1994, Viral Immunol. 7:25-30, and viral titers in lunghomogenates were determined using an MDCK cell-chicken RBChemagglutination assay.

Cytotoxic Assay. A primary cytotoxicity assay was carried out byincubating effector cells with 5×10³ ⁵¹Cr-labeled target cells atdifferent effector-to-target ratios in 96-well V-bottom plates. P815target cells were infected with PR8 virus for 1 hour before labelingwith ⁵¹Cr or incubated during the assay with 5-10 ∥g/ml of NP₁₄₇₋₁₅₅.After incubation for 4 hours at 37° C. in 5% CO₂, the supernatant washarvested and radioactivity released was determined using a gammacounter. A secondary cytotoxicity assessment was carried out afterco-culturing equal numbers of lymphocytes from test animals andx-irradiated, virus-infected or NP₁₄₇₋₁₅₅-coated lymphocytes fromnon-immunized BALB/c mice for five days in RPMI supplemented with fetalcalf serum (“FCS”) 10% and 50 μM 2-mercaptoethanol; the secondary CTLassay itself was conducted using the ⁵¹Cr release assay described above,and the results were expressed as the percentage of specific lysisdetermined in triplicate for each effector:target ratio employed, asfollows:

100(actual−spontaneous release)÷(maximum−spontaneous release−backgroundrelease)±SD

Limiting Dilution Analysis of CTL Precursors. The number ofantigen-specific CTL precursors in the spleens of immunized mice wereassessed by incubating single-cell suspensions of splenic respondercells in six steps of two-fold dilutions with 2.5×10⁵ X-irradiated,PR8-infected syngeneic splenocytes. After five days in complete RPMImedium, individual microtiter cultures were assayed using ⁵¹Cr releasefrom P815 cells infected with influenza virus; uninfected P815 cellswere used as a control. Those wells exhibiting ⁵¹Cr release greater thanbackground plus three standard deviations were regarded as positive. Thepercentage of cultures in one dilution step regarded as negative forspecific cytotoxicity were plotted logarithmically against the number ofresponder cells/well, and the frequency of CTL precursors was determinedby linear regression analysis using the following formula:

−1n(negative-well index)÷(number of responder cells/well)=1/(number ofresponder cells/well at 0.37 negative well index).

The number of precursor cells is represented as 1/frequency for purposesof comparison.

Plasmid Detection by PCR. Injected and control muscle tissue was removedone month after completion of the vaccination schedule, immediatelyfrozen in ethanol-dry ice, and stored at −80° C. Frozen tissue washomogenized in lysis buffer and DNA was extracted as described inMontgomery, 1993, DNA and Cell Biol. 12:777-783 and Ulmer et al., 1993,Science 254:1745. A forty-cycle PCR reaction was carried out withNP-specific primers located at the following nucleotide positions: 1120(minus strand; 5′-[CATTGTCTAGAATTTGAACTCCTCTAGTGG]-3′; SEQ ID NO:18;Cerbone) as well as 468 (positive strand; 5′-[AATTTGAATGATGCAAC]-3′; SEQID NO:20). A PCR product with a specific signal of 682 bp was visualizedusing ethidium bromide stained agarose gels.

Hemagglutination Inhibition Assay. Sera from immunized mice were treatedwith receptor destroying enzyme (RDE/neuraminidase) for 1 hour at 37° C.in a waterbath. Two-fold serial dilutions of RDE-treated sera wereincubated with 0.5% human erythrocyte saline suspension in the presenceof hemagglutinating titers of influenza virus. The experiment wascarried out in triplicate wells. After 45 minutes incubation in a96-well round bottom RIA plates (Falcon) at room temperature, theresults were read and expressed as log₂ of the last inhibitory dilution.Negative controls (blank sera) and positive controls (HA specificmonoclonal antibodies) were included in the experiment.

Cytokine Measurement by ELISA. T cells were incubated, for four days,with antigen and irradiated accessory cells, and then 100 microliters ofsupernatant were harvested from each microculture. The concentrations ofIFN gamma and IL-4 were measured using ELISA test kits (Cytoscreen, fromBiosource Int. and Interest from Genzyme, respectively). Standards withknown concentrations were included in the assay. The optical densitieswere assessed at 450 nm absorbance after blanking the ELISA read on thenull concentration wells.

6.2. Results

Priming of CTL Precursors via Neonatal DNA Vaccination. The optimalschedule for DNA vaccination in the experiments described was developedin pilot studies. Newborn mice were immunized with 30 μg of NPV1 orcontrol plasmid on Days 1, 3 and 6 after birth; adult animals werevaccinated with the same amount of DNA immunogen on Days 0, 21 and 42 ofthe study. One month after the completion of this standard series ofvaccinations, certain test animals were boosted with live PR8 virus.

The lymphocytes, directly isolated from newborn and adult micevaccinated with NPV1 and boosted with PR8 virus, lysed target cellscoated in vitro with NP₁₄₇₋₁₅₅, which is recognized by CTL inassociation with K^(d) MHC-molecules of Class I (FIGS. 1A-F). No primarycytotoxicity was observed in vitro with lymphocytes from newbornsimmunized on Day 1 with PR8 virus and boosted one month later with PR8virus. As expected, significant cytotoxicity was observed after in vitroexpansion of splenocytes from mice immunized with NP-V1 plasmid or PR8virus only. No significant cytotoxicity was observed in the case of miceimmunized with control virus or B/Lee virus. These data clearly indicatethat vaccination with NPV1 with or without subsequent boosting withnative virus induced an expansion of NP-specific CTL precursors in bothnewborn animals and adults; however, both primary cytotoxicity andimmunologically significant secondary cytotoxicity were observed only inanimals fully immunized with NPV1 and boosted with virus.

Frequency of NP-specific CTL Precursors. An immunologically significantincrease in the frequency of NP-specific CTL precursors was observed inanimals immunized with NPV1 and boosted with PR8 virus, accounting forthe presence of primary cytotoxicity in this particular group (FIGS.2A-B). The increased frequency of specific precursors is presumably dueto sustained biosynthesis of NP antigen, which primed and expanded thispopulation of NP-specific lymphocytes. Plasmid was detected byqualitative PCR one month after completion of the immunization series ingluteal muscle, the site of injection of NPV1 in newborns, and in tibialmuscle, the site of injection of NPV1 in adult animals (FIG. 3).

Induction of Cross-reactive CTLs via DNA Immunization. The induction ofcross-reactive CTLs against NP-subtypes in adult animals immunized withtype A influenza virus is well-characterized and understood to berelated to the limited genetic variation of NP compared to hemagglutinin(HA) and neuraminidase (NA), which are viral surface proteins. In asimilar manner, CTLs derived from newborn mice immunized with NPV1 andboosted with PR8 virus exhibited increased lysis of P815 cells infectedwith a variety of influenza strains, including PR8(H₁N₁), A/HK/68(H₃N₂)and A/Japan(H₂N₂) viruses, but not the Type B virus B/Lee, after invitro stimulation with PR8 virus infected cells or NP₁₄₇₋₁₅₅ peptide(FIGS. 4A-C).

Effect of DNA Immunization on Pulmonary Virus Titer. The increasedactivity of CTLs in those animals vaccinated with NPV1 is correlatedwith decreased viral titers in lung tissue measured after aerosolchallenge with one LD₁₀₀ of PR8 or HK viruses. Although no difference inviral titers was observed in mice immunized with NPV1 or control plasmidthree days after PR8 challenge, a statistically significant reductionwas observed in both newborn (p<0.05) and adult mice (p<0.025; Table I)seven days after challenge. No virus was detected in the lungs of micethat survived challenge for more than 16 days. It is important to notethat decreased viral titers in lung tissue were observed in micechallenged with PR8 virus one or three months after completing theimmunization (p<0.05).

Effect of DNA Immunization on Clinical Course of Infection and Survival.Genetic immunization of adult mice with NPV1 induced protective immunityin 80% of animals challenged with PR8 virus one month after the lastimmunization (p<0.01; FIGS. 5A-D) and in 57 percent of adult animalschallenged three months after the last immunization (p<0.05; FIG. 5E).An increased survival after challenge was observed in three month oldmice immunized with NPV1 as newborns, indicating that during the threemonth period a more vigorous expansion of CTL precursors was elicitedafter genetic immunization (p<0.02, FIG. 5B). Only 10% of adult animalschallenged with HK virus survived (FIGS. 5A-D), findings that differfrom those previously reported (Ulmer et al., 1993, Science259:1745-1749) even though the DNA immunogen and HK strain used inchallenge were identical. The relative decrease in survival we observedcould be explained by the intranasal route of challenge used previously(Id.), which is less likely to provide productive infection of the loweras well as upper respiratory tract compared to the aerosol challengeemployed in these studies. Despite their immunoresponsiveness, one-monthold mice immunized with NPV1 as newborns exhibited reduced survivalafter challenge with PR8 and no survival after challenge with HK viruscompared to immunized adults and three month old mice infected with NPV1as newborns, which exhibited significant survival after challenge withLD100 of PR8 virus.

The pneumonia that occurs after influenza infection is accompanied byweight loss in these animals. Adult mice treated with control plasmidand challenged with a lethal dose of PR8 gradually lost weight untilthey expired (Days 7-9), while the surviving animals immunized with NPV1recovered their prechallenge body weight by Day 10 after significantinitial weight loss post-challenge (Day 2-7; FIGS. 6A-B). Similarresults were obtained with one-month old mice which had been immunizedafter birth as newborns with NPV1 (FIGS. 6A-B) or with three month oldmice.

Effect of DNA Immunization with a Plasmid Which Encodes HA of InfluenzaVirus (pHA plasmid). Immunization of newborn mice with pHA according tothe same protocol as NPV1 was followed by specific antibody productionas early as 1 month after birth which persisted at least three monthsafter birth (Table 2). These antibodies displayed hemagglutinationinhibiting properties, like antibodies obtained by live-virus or plasmidimmunization of adult mice. In consequence, immunization of neonateswith pHA elicited protective, virus-specific antibodies.

Immunization of mice with pHA primed T helper cells which were then ableto secrete cytokines upon in vitro restimulation with virus (Table 3).Whereas pHA injection of adult mice elicited predominantly TH1 typecells, inoculation of neonates with the same plasmid lead to thedevelopment of a mixed Th1/Th2 response. DNA immunization of neonates aswell as adult mice with pHA conferred significant protection to lethalchallenge (LD₁₀₀) with WSN or PR8 virus as early as one month afterimmunization (FIGS. 7A-D).

6.3. Discussion

Numerous studies have indicated that the genetic immunization of adultmice, chickens, ferrets and monkeys with cDNAs containing NP or HAsequences of various strains of type A influenza virus can induceprotective cellular and humoral immunity (Ulmer et al., 1993, Science258:1745-1749; Montgomery et al., 1993, DNA and Cell Biol. 12:777-783;Fyneu et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:11478-11482;Justevicz et al., 1995, J. Virol. 19:7712-7717; Donnely et al., 1995,Nature Med. 1:583-587). The results presented herein are the firstevidence that such immunization has a comparable effect in newbornanimals, and that cellular immunity is generated consequent to a strongpriming effect characterized by a significant increase in the frequencyof antigen-specific CTL precursors. The survival after challenge, thereduction in viral lung titers and recovery of prechallenge body weightcompared to controls in animals that were vaccinated with NPV1 or pHA isindicative of effective secondary immune responses.

Previous studies in adult mice have indicated that immunization withhomologous virus affords 100% protection to lethal challenge, while only50-60% protection occurs in normal mice infused with NP-specific T cellclones (Taylor et al., 1986, Immunology 58:417-420) or in PR8-immunizedB cell deficient (J_(H)D−/−) animals (Bot et al., 1996, J. Viroo.70:5668-5672), indicating that effective protection requires bothhumoral and cellular responsiveness, the former presumably mitigatingthe spread of virus and the extent of pulmonary lesions. The absence ofa protective antibody response in the studies carried out with NPV1plasmid as well as slow expansion of CTL precursors during the firstmonth of life may explain the relatively poor survival of one month oldmice that were immunized with NPV1 plasmid as newborns. The increasedsurvival of three month old mice immunized as newborns with NPV1 plasmidsuggests that the expansion of CTL precursors continues after neonatalimmunization, enabling the mice to develop a stronger cellular responsewhen they become adults.

Further data indicates that the plasmid expressing the HA gene of WSNvirus, injected after birth, elicits both humoral and cellular responsesmirrored in an increased survival. For example, neonatal immunizationwith pHA triggered an antibody response associated with a helperresponse which conferred significant protection upon later challengewith influenza virus.

TABLE 1 Effect of immunization with NPV1 plasmid on pulmonary virustiter measured after challenge with lethal doses of PR8 or HK virus.challenge with challenge with 1.5 × 10⁴ 3.2 × 10⁵ age of TCID₅₀ PR8virus TCID₅₀ HK virus animals immunization 3 d 7 d 16 d 3 d 7 d 16 dadult nil 4.6 ± 3.8 ± +³ 6.4 ± 5.7 ± + 0.5 0.1 0.7 0.3 PR8 virus 0 0 ND5.7 ± 0 ND 0.3 control plasmid 4.8 ± 3.7 ± + 6.8 ± 5.7 + 0.1 0.5 0.1NPV1-1 month¹ 4.0 ± 0.9 ± 0⁴ 5.8 ± 0.6 ± 0 0.3 1.5 0.1 1.1 NPV1-3months² 4.8 ± 0.2 ± 0 6.9 ± 4.6 ± + 0.1 0.2 0.7 0.8 newborn controlplasmid 5.9 ± 4.6 ± + ND ND ND 0 0.2 NPV1-1 month 4.5 ± 1.2 ± 0 6.6 ±5.1 ± + 1.2 2.1 0.3 0.6 NPV1-3 months 4.1 ± 0.9 ± 0 ND ND ND 0.5 1.2Mice were sacrificed 1 month after the last immunization. Data areexpressed as log₁₀ of viral titer in TCID₅₀ units. ND—not done ¹micechallenged 1 month after completing the immunization ²mice challenged 3months after completing the immunization ³no survivors at day 16 afterchallenge ⁴pulmonary virus titer in mice which survived more than 16days

TABLE 2 HI titer of BALB/c mice immunized with WSN virus or plasmidsMice Prebleeding No. of Titer 7 days after immunized Immunization No. oftiter Time of Titer against: responders boost against: as: with: mice:WSN PR8 bleeding: WSN PR8 WSN PR8 Boost: WSN PR8 Adults WSN 5  0^(a) 0 1mo  8.2 ± 1.1^(b) 1.2 ± 0.8 5/5 5/5 WSN 8.2 ± 1.3 2.2 ± 1.6 CP 3 0 0 1mo 0 1.0 ± 0.7 0/3 1/3 — 0 0 CP 3 0 0 1 mo 0 0 0/3 0/3 WSN 7.3 ± 5.3 1.3± 2.3 pHA 16  0 0 1 mo 5.5 ± 3.4 0 12/16  0/16 WSN 8.3 ± 1.5 1.0 ± 1.9pHA 8 0 0 3 mo 8.7 ± 3.8 0 5/8 0/8 WSN 8.3 ± 1.5 2.0 ± 2.0 pHA 9 0 0 6mo 1.0 ± 0   0 2/9 0/9 WSN 8.3 ± 0.6 1.3 ± 0.6 pHA 3 0 0 9 mo 0 0 0/30/3 WSN 5.6 ± 0.6 5.0 ± 1.7 Newborns CP 5 ND ND 1 mo 0 0 0/5 0/5 WSN 7.0± 0.8 0 pHA 19  ND ND 1 mo 5.2 ± 2.7 0 12/19  0/19 WSN 9.4 ± 0.9 2.0 ±1.6 pHA 4 ND ND 3 mo 3.3 ± 1.5 0 3/4 0/4 WSN 8.8 ± 2.9 3.2 ± 2.5 ^(a)0 =<1:40 ^(b) log₂ dilution ND—not done

TABLE 3 Table Lymphokine production by T cells from mice immunized withpHA plasmid or WSN virus: Group Immun- Lymph- Adult mice Newborn miceization Boost kines nil* WSN* nil WSN nil — IFNγ 0 0 ND ND — IL-4 0 0 NDND CP — IFNγ 0  11 ± 5** 14 ± 5  22 ± 3  — IL-4 0 0 0 0 WSN IFNγ 24 ± 1 158 ± 4  89 ± 28 261 ± 26  WSN IL-4 236 ± 11  79 ± 19 198 ± 5  141 ± 39 pHA — IFNγ 9 ± 1 60 ± 2  0 29 ± 18 — IL-4 0 0 2 ± 2 6 ± 3 WSN IFNγ 19 ±3  284 ± 10  38 ± 8  179 ± 50  WSN IL-4 54 ± 3  31 ± 4  138 ± 4  257 ±24  WSN — IFNγ 52 ± 2  214 ± 11  103 ± 30  51 ± 8  — IL-4 48 ± 3  181 ±3  132 ± 6  248 ± 20  WSN IFNγ 10 ± 1  127 ± 3  9 ± 5 61 ± 12 WSN IL-4218 ± 4  235 ± 12  228 ± 8  594 ± 5  *1.5 × 10⁵ nylon wool non-adherentsplenocytes were incubated for four days with 1.5 × 10⁵ irradiatedBALB/c splenocytes with or without 10 μg/ml UV-innactivated WSN virus,in presence of 1 U/ml exogenous IL-2. **concentration of cytokines insupernatant was determined by ELISA and expressed as pg/ml. Values belowbackground ± 3 × SD were considered 0.

Various publications are cited herein, the contents of which areincorporated by reference in their entireties.

20 19 amino acids amino acid single linear peptide unknown 1 Arg Lys SerIle His Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr Gly 1 5 10 15 Glu IleIle 10 amino acids amino acid single linear peptide unknown 2 Trp LeuThr Lys Lys Gly Asp Ser Tyr Pro 1 5 10 10 amino acids amino acid singlelinear peptide unknown 3 Trp Leu Thr Lys Ser Gly Ser Thr Tyr Pro 1 5 1010 amino acids amino acid single linear peptide unknown 4 Trp Leu ThrLys Glu Gly Ser Asp Tyr Pro 1 5 10 11 amino acids amino acid singlelinear peptide unknown 5 Ile Asn Gln Asp Pro Asp Lys Ile Leu Thr Tyr 1 510 19 amino acids amino acid single linear peptide unknown 6 Met Asn SerAla Pro Asn Leu Arg Gly Asp Leu Gln Lys Val Ala Arg 1 5 10 15 Thr LeuPro 11 amino acids amino acid single linear peptide unknown 7 Ser PheGlu Arg Phe Glu Ile Phe Pro Lys Glu 1 5 10 20 amino acids amino acidsingle linear peptide unknown 8 Asn Ser Val Asp Asp Ala Leu Ile Asn SerThr Lys Ile Tyr Ser Tyr 1 5 10 15 Phe Pro Ser Val 20 17 amino acidsamino acid single linear peptide unknown 9 Pro Glu Ile Asn Gly Lys AlaIle His Leu Val Asn Asn Glu Ser Ser 1 5 10 15 Glu 15 amino acids aminoacid single linear peptide unknown 10 Ala Asn Glu Arg Ala Asp Leu IleAla Tyr Leu Gln Ala Thr Lys 1 5 10 15 20 amino acids amino acid singlelinear peptide unknown 11 Asp Gln Val His Phe Gln Pro Leu Pro Pro AlaVal Val Lys Leu Ser 1 5 10 15 Asp Ala Leu Ile 20 14 amino acids aminoacid single linear peptide unknown 12 Asp Gly Ser Thr Asp Tyr Gly IleLeu Gln Ile Asn Ser Arg 1 5 10 12 amino acids amino acid single linearpeptide unknown 13 Gln Val Glu Lys Ala Leu Glu Glu Ala Asn Ser Lys 1 510 20 amino acids amino acid single linear peptide unknown 14 Arg ThrAsp Lys Tyr Gly Arg Gly Leu Ala Tyr Ile Tyr Ala Asp Gly 1 5 10 15 LysMet Val Asn 20 15 amino acids amino acid single linear peptide unknown15 Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp Pro 1 5 10 1515 amino acids amino acid single linear protein unknown 16 Ile Ala SerAsn Glu Asn Met Asp Ala Met Glu Ser Ser Thr Leu 1 5 10 15 9 amino acidsamino acid single linear protein unknown 17 Lys Ala Val Tyr Asn Phe AlaThr Met 1 5 8 amino acids amino acid single linear protein unknown 18Ser Ile Ile Asn Phe Glu Lys Leu 1 5 30 bases nucleic acid single linearcDNA unknown 19 CATTGTCTAG AATTTGAACT CCTCTAGTGG 30 17 bases nucleicacid single linear cDNA unknown 20 AATTTGAATG ATGCAAC 17

What is claimed is:
 1. A method for immunizing an infant mammal againsta target viral antigen, comprising inoculating the mammal, while aninfant, with an effective amount of a naked recombinant nucleic acidmolecule encoding a peptide comprising one or more relevant epitopes ofthe target viral antigen in a pharmaceutical carrier, such that atherapeutically effective amount of the relevant peptide is expressed inthe infant mammal, wherein said infant is immunized.
 2. The method ofclaim 1, wherein the target antigen is a respiratory syncytial virusantigen.
 3. The method of claim 1, wherein the target antigen is arotavirus antigen.
 4. The method of claim 1, wherein the target antigenis a measles virus antigen.
 5. The method of claim 1, wherein the targetantigen is a human immunodeficiency virus antigen.
 6. The method ofclaim 1, wherein the target antigen is a hepatitis virus antigen.
 7. Themethod of claim 1, wherein the target antigen is a hepatitis B virusantigen.
 8. The method of claim 1, wherein the target antigen is aherpes simplex virus antigen.
 9. The method of claim 1, wherein thetarget antigen is an influenza virus antigen.
 10. The method of claim 1wherein maternal antibodies are present in detectable amounts in theinfant mammal.
 11. The method of claim 1 wherein the mammal is a humanhaving an age extending from birth to the age of nine months.
 12. Themethod of claim 1 wherein the mammal is a human having an age extendingfrom birth to the age of one month.
 13. The method of claim 1 whereinthe infant mammal is a neonate.
 14. A method for immunizing an infantmammal against a target viral antigen, comprising injection into themammal of an effective amount of a naked nucleic acid molecule encodinga viral peptide comprising the target antigen comprising one or morerelevant viral epitopes in a pharmaceutically acceptable carrier, suchthat a therapeutically effective amount of the relevant peptide isexpressed in the infant mammal, wherein said infant is immunized.
 15. Amethod for immunizing an infant mammal against a target viral antigen,comprising inoculating the mammal with a therapeutically effectiveamount of a naked recombinant nucleic acid molecule encoding a peptidecomprising one or more relevant viral epitopes of the target viralantigen in a pharmaceutical acceptable carrier, wherein; (i) thetherapeutical effective amount of nucleic acid is introduced by aplurality of inoculations all administered while the animal is aninfant; and (ii) immunization results in significant resistance to adisease associated with a pathogen that expresses the target antigen.16. The method of claim 15, wherein the mammal is a human.
 17. Themethod of claim 15, wherein the mammal is a human and the first of theplurality of injections is administered at an age extending form birthto about six months.
 18. The method of claim 15, wherein the mammal is ahuman and the first of the plurality of injections is administered at anage extending from birth to about one month.
 19. The method of claim 15,wherein the mammal is a human and the first of the plurality ofinjections is administered at an age extending from birth to about oneweek.